fos/jun repression of cardiac-specific transcription in quiescent and growth-stimulated myocytes is targeted at a tissue-specific cis element.

Unlike that of skeletal muscle cells in which growth and differentiation appear mutually exclusive, growth stimulation of cardiac cells is characterized by transient expression of early response nuclear proto-oncogenes as well as induction of several cardiac-specific markers. This observation led to the speculation that these proto-oncogenes, particularly c-fos and c-jun, might act as positive regulators of cardiac transcription. We have examined the role of c-jun and c-fos in basal and growth-stimulated cardiac transcription, using the cardiac-specific atrial natriuretic factor (ANF) gene as a marker. The results indicate that c-jun and c-fos are negative regulators of ANF transcription. Inducers of jun and fos activity, such as mitogens and growth factors, inhibited endogenous ANF transcripts. In transient cotransfection assays, jun and fos were able to trans-repress the ANF promoter in both quiescent and alpha 1-adrenergic stimulated myocytes. This repression was specific to myocyte cultures and was not observed in nonmuscle cells. Deletion analysis indicated that repression does not require typical AP-1-binding sites (tetradecanoyl phorbol acetate response elements) or serum response elements but is targeted at a cardiac-specific element within the ANF promoter. Various Fos-related proteins, including Fra-1, Fos B, and v-Fos, were able to trans-repress ANF transcription. In addition, C-terminal c-fos mutants which no longer repress transcription of such early growth response genes as c-fos and EGR-1 retained the ability to repress ANF transcription. Repression by c-jun occurs via the N-terminal activation domain and does not require the DNA-binding domain, suggesting that proto-oncogene repression involves interaction with one or more limiting cardiac-specific coactivators.

Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells.

Cardiac myocytes undergo a major genetic switch within the first week of postnatal development, when cell division ceases terminally and many cardiac genes are either activated or silenced. We have developed stage-specific cardiocyte cultures to analyze transcriptional control of the rat atrial natriuretic factor (ANF) gene to identify the mechanisms underlying tissue-specific and developmental regulation of this gene in the heart. The first 700 bp of ANF flanking sequences was sufficient for cardiac muscle- and stage-specific expression in both atrial and ventricular myocytes, and a cardiac muscle-specific enhancer was localized between -136 and -700 bp. Deletion of this enhancer markedly reduced promoter activity in cardiac myocytes and derepressed ANF promoter activity in nonexpressing cells. Two distinct domains of the enhancer appeared to contribute differentially to cardiac specificity depending on the differentiation stage of the myocytes. DNase I footprinting of the enhancer domain active in differentiated cells revealed four putative regulatory elements including an A+T-rich region and a CArG element. Deletion mutagenesis and promoter reconstitution assays revealed an important role for the CArG-containing element exclusively in cardiac cells, where its activity was switched on in differentiated myocytes. Transcriptional activity of the ANF-CArG box correlated with the presence of a cardiac- and stage-specific DNA-binding complex which was not recognized by the c-fos serum response element. Thus, the use of this in vitro model system representing stage-specific cardiac development unraveled the presence of different regulatory mechanisms for transcription of the ANF gene during cardiac differentiation and may be useful for studying the regulatory pathways of other genes that undergo switching during cardiac myogenesis.

Relation between preferred and optimal cadences during two hours of cycling in triathletes.

OBJECTIVES: To determine whether the integrated electromyographic signal of two lower limb muscles indicates preferred cadence during a two hour cycling task. METHODS: Eight male triathletes performed right isometric maximum voluntary contraction (MVC) knee extension and plantar flexion before (P1) and after (P2) a two hour laboratory cycle at 65% of maximal aerobic power. Freely chosen cadence (FCC) was also determined, also at 65% of maximal aerobic power, from five randomised three minute sessions at 50, 65, 80, 95, and 110 rpm. The integrated electromyographic signal of the vastus lateralis and gastrocnemius lateralis muscles was recorded during MVC and the cycle task. RESULTS: The FCC decreased significantly (p<0.01) from P1 (87.4 rpm) to P2 (68.6 rpm), towards the energetically optimal cadence. The latter did not vary significantly during the cycle task. MVC of the vastus lateralis and gastrocnemius lateralis decreased significantly (p<0.01) between P1 and P2 (by 13.5% and 9.6% respectively). The results indicate that muscle activation at constant power was not minimised at specific cadences. Only the gastrocnemius lateralis muscle was affected by a two hour cycling task (especially at 95 and 110 rpm), whereas vastus lateralis remained stable. CONCLUSION: The decrease in FCC observed at the end of the cycle task may be due to changes in the muscle fibre recruitment pattern with increasing exercise duration and cadence.

Endurance and strength training effects on physiological and muscular parameters during prolonged cycling.

PURPOSE: This study investigated the effects of a combined endurance and strength training on the physiological and neuromuscular parameters during a 2-h cycling test. METHODS: Fourteen triathletes were assigned to an endurance-strength training group and an endurance-only training group. They performed three experimental trials before and after training: an incremental cycling test to exhaustion, a maximal concentric lower-limbs strength measurement and a 2-h cycling exercise. Physiological parameters, free cycling chosen cadence and the EMG of Vastus Lateralis (VL) and Rectus Femoris (RF) were analysed during the 2-h cycling task before and after a strength training programme of 5 weeks (three times per week). RESULTS: The results showed that the maximum strength and the isometric maximal voluntary contraction (isoMVC) after training were significantly higher (P<0.01) and lower (P<0.01) than those before training, respectively, in endurance-strength training group and endurance-only group. The physiological variables measured during the cycling tests and the progressive increase (P<0.01) in EMGi(VL) and EMGi(RF) throughout the 2-h cycling test did not differ between the two groups before and after training, except for the variation of EMGi(VL) over the cycle time which was stabilized during the second hour of the 2-h cycling test due to training in endurance-strength training group. The decrease in free cycling chosen cadence observed in pre-training (P<0.01) was also replaced by a steady free cycling chosen cadence for the endurance-strength training group during the second hour of exercise. CONCLUSION: This study confirmed the decrease in the free cycling chosen cadence with exercise duration and demonstrated that a specific combined endurance and strength training can prevent this decrease during a 2-h constant cycling exercise.

Thyroid hormone stimulates rat pro-natriodilatin mRNA levels in primary cardiocyte cultures.

Pro-natriodilatin (PND) is the precursor for atrial natriuretic peptide (ANP), a hormone which plays an important role in cardiovascular homeostasis. Since the effects of thyroid hormone (T3) on the cardiovascular and renal systems appear to mimic those elicited by ANP, we studied the effect of T3 on PND gene expression using rat neonatal cardiocytes in primary cultures. Treatment of cardiocytes for 48 h with T3 (5 X 10(-9) M) results in a maximal increase in PND mRNA levels; this increase is two fold in atrial and four fold in ventricular cell cultures. These results taken together with a previous report showing decreased plasma ANP in hypothyroid and increased plasma ANP in hyperthyroid rats suggest that at least some of the cardiovascular and renal effects of T3 may be mediated by a T3-dependent increase in PND gene expression.

Distal cis-acting promoter sequences mediate glucocorticoid stimulation of cardiac atrial natriuretic factor gene transcription.

Although receptors for most steroid hormones are present in the heart, few cardiac-specific target genes have been identified and studied at the molecular level. Transcription of the atrial natriuretic factor (ANF) gene, which encodes the major secretory product of the heart, is induced by glucocorticoids. In both atrial and ventricular cardiac cells in primary cultures, ANF mRNA levels are increased 3-4-fold after dexamethasone treatment in a time- and dose-dependent manner. This response to glucocorticoids is completely abolished by the antagonist RU486. Interestingly, ventricular myocytes appear to be more sensitive to glucocorticoids than atrial myocytes. DNA-mediated gene transfer studies indicate that glucocorticoids affect ANF gene transcription via a glucocorticoid response element located in the distal 5'-flanking sequences of the rat ANF gene between -697 and -1,029 base pairs. In vitro DNase I footprinting experiments reveal the presence of two binding sites for purified glucocorticoid receptor within this region. Mobility shift assays and competition experiments show that binding of the glucocorticoid receptor to both ANF sites results in a DNA-protein complex similar in affinity and specificity to that of the well characterized mammary tumor virus glucocorticoid response element. Since glucocorticoid activation of the ANF promoter appears specific to cardiac cells, the interaction between the glucocorticoid receptor binding sites and cardiac-specific regulatory elements of this promoter could provide a model to study a mechanism of hormone-dependent signal transduction in the heart.

Localization of the human B-type natriuretic peptide precursor (NPPB) gene to chromosome 1p36.

Cardiac myocytes synthesize and secrete a family of peptide hormones with potent natriuretic, diuretic, and vasodilatory properties. These peptides are derived from precursor molecules that are encoded by two different genes, the atrial natriuretic peptide precursor A (NPPA) and the B-type natriuretic peptide or natriuretic peptide precursor B (NPPB). A human genomic clone for the NPPB gene was used to determine the chromosomal location of the NPPB gene. Analysis of Southern blot hybridization to DNAs from various somatic cell hybrids and fluorescence in situ hybridization allowed assignment of the NPPB locus to human chromosome 1p36. This location coincided with that of the NPPA locus; pulsed-field gel electrophoresis placed NPPA and NPPB within 50 kb of each other. This close chromosomal linkage, together with the conserved primary sequences and structural organization of the two natriuretic peptide precursor genes, suggests that the natriuretic peptide loci may have evolved from a common ancestor gene.

Myocardial performance index as predictor of adverse outcomes following mitral valve surgery.

AIMS: We aim to determine whether the myocardial performance index, will be a good predictor of adverse outcomes following mitral valve surgery. METHOD: We prospectively measured pre-operative myocardial performance index in 22 consecutive patients, with moderate to severe mitral insufficiency, undergoing corrective mitral valve surgery. The primary endpoint was predefined as either peri-operative death or congestive heart failure. RESULTS: The primary endpoint occurred in nine patients. Five of the six patients with myocardial performance index >or=0.7 had primary endpoints. Chi-square testing demonstrated that the primary endpoint was significantly associated with advanced age (>or=70 years) and myocardial performance index >or=0.7 (P=0.003 and 0.01 respectively). There was a trend towards significant association of depressed left ventricle ejection fraction (left ventricle ejection fraction or=0.7. CONCLUSION: Our results suggest that myocardial performance index is a potentially useful predictor of increased risk of peri-operative death or congestive heart failure, in patients with moderate-severe mitral insufficiency undergoing corrective mitral valve surgery. In conjunction with left ventricle ejection fraction, it may be helpful in the pre-operative prognostication of these patients.

Cloning and expression of the atrial natriuretic factor gene.

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.

The gene for rat atrial natriuretic factor.

Atrial natriuretic factor (ANF), a peptide hormone recently isolated from heart atria, appears to play an important role in the regulation of extracellular fluid volume and blood pressure. Indeed, natural and synthetic ANF rapidly and markedly stimulate natriuresis and diuresis and produce smooth muscle relaxation. Consistent with the hypothesis that ANF is a novel hormone, it was recently shown that ANF is present in circulation, and high affinity membrane receptors specific for ANF have been described in renal, vascular, and adrenal tissues. These important biological activities suggest that conditions like hypertension could be associated with defective ANF gene expression. We and others have shown by cDNA cloning that ANF is part of a larger precursor, pro-natriodilatin (PND). We now describe the isolation and structural analysis of the rat PND gene. Southern blot analysis of rat genomic DNA suggests the presence of a single PND gene per haploid genome. The PND coding sequences are interrupted by two short introns. A long alternating purine-pyrimidine tract (GT)9GATG(GT)27 is found 111 base pairs downstream of the polyadenylation site; such sequences could adopt Z-DNA configuration and they have been associated with sequences that appear very active in intergenic recombination. Comparison of the rat and human PND genomic sequences shows highest homology in 5'-flanking as well as in coding sequences. The rat PND gene will be a useful model to study the physiology and pathology of this important regulator of the cardiovascular system.

Gene structure of human cardiac hormone precursor, pronatriodilatin.

Atrial cardiocytes contain granules typical of protein-secreting cells, and atrial extracts are known to contain a powerful natriuretic and diuretic activity and to possess smooth muscle relaxant activity. A variety of active atrial peptides have been isolated, including a family of related peptides showing natriuretic, diuretic and smooth muscle relaxant activities in rat and human atria; these peptides were named atrial natriuretic factor (ANF). Another unrelated peptide from pig atria, cardiodilatin, is thought to possess only smooth muscle relaxant activity. Its partial amino acid sequence shows no homology with ANF sequences. The sequence analysis of a large form (106 amino acids) of ANF and of ANF complementary DNA clones indicates that cardiodilatin and ANF peptides are synthesized from a common precursor. This precursor also contains a signal peptide sequence expected of a secretory protein. We now describe the complete structure and sequence of the human gene for this novel hormone precursor that we call pronatriodilatin.